Hisao Takizawa

Direct Assessment of MHC Class I Binding by Seven Ly49 Inhibitory NK Cell Receptors

Mouse NK cells express at least seven inhibitory Ly49 receptors. Here we employ a semiquantitative cell-cell adhesion assay as well as class I/peptide tetramers to provide a comprehensive analysis of specificities of Ly49 receptors for class I MHC molecules in eight MHC haplotypes. Different Ly49 receptors exhibited diverse binding properties. The degree of class I binding was related to the extent of functional inhibition. The tetramer studies demonstrated that neither glycosylation nor coreceptors were necessary for class I binding to Ly49 receptors and uncovered peptide-specific recognition by a Ly49 receptor. The results provide a foundation for interpreting and integrating many existing functional studies as well as for designing tests of NK cell development and self-tolerance.

Clonal Acquisition of Inhibitory Ly49 Receptors on Developing NK Cells Is Successively Restricted and Regulated by Stromal Class I MHC

We report an in vitro stroma-dependent system for the clonal growth and differentiation of natural killer (NK) cells from lymphoid-restricted bone marrow progenitors or bone marrow NK1.1+ cells. Strikingly, the potential to initiate expression of specific Ly49 receptors becomes increasingly restricted as NK cells develop. Moreover, when NK cells express a Ly49 receptor specific for stromal cell class I MHC, they are less likely to initiate expression of another Ly49 receptor in the clonal culture system. The results indicate multiple roles for stromal cells in NK cell development, in supporting clonal growth, in initiation of Ly49 receptor expression, and in formation of the NK cell receptor repertoire.

Memory CD8 T lymphocytes express inhibitory MHC-specific Ly49 receptors

Natural killer (NK) cells survey potential targets using an array of receptors specific for major histocompatibility complex class I molecules. In mice, members of the Ly49 receptor gene family are expressed on overlapping subsets of NK cells and on CD1‐restricted NK1 T cells. Here we characterize a population of memory cytotoxic (CD8+) T lymphocytes which also express inhibitory Ly49 family members. This cell population increases steadily with age; by 11 months, over one third of memory CD8+ T cells express Ly49 molecules. These cells appear to express a normal TCR repertoire, and share several traits with previously activated T cells. Analysis of mutant mouse strains reveals that normal development of these cells depends upon the presence of the transporter associated with antigen presentation (TAP), classical class I molecules, and class II molecules. As a functional consequence of Ly49 expression, we demonstrate that T cell receptor‐mediated activation of CD8+ T cells is inhibited by Ly49 interactions with cognate class I molecules. We hypothesize that conventional memory CD8+ T cells initiate Ly49 expression as a means of dampening an immune response and / or inhibiting T cell autoreactivity.

The Fab Fragment of a Novel Anti-GPVI Monoclonal Antibody, OM4, Reduces In Vivo Thrombosis Without Bleeding Risk in Rats

Background—Inhibition of GPVI has been proposed as a useful antithrombotic strategy; however, in vivo proof-of-concept animal studies targeting GPVI are lacking. We evaluated a novel anti-human GPVI monoclonal antibody OM4 Fab in rats. Methods and Results—OM4 Fab specifically inhibited collagen-induced aggregation of rat platelets in vitro with an IC50 of 20 to 30 &mgr;g/mL but not ADP and AA-induced platelet aggregation. After intravenous administration of OM4 Fab, a rapid inhibition of ex vivo platelet aggregation was observed with a gradual recovery within 60 to 90 minutes which corresponded to the decline in OM4 Fab plasma concentration and time-dependent decrease in platelet-bound OM4 Fab. In contrast to previous reports in mice, intravenous OM4 Fab did not deplete platelet GPVI. Injection of OM4 IgG caused acute thrombocytopenia. In a modified Folts model of cyclic flow reduction in rat carotid artery, the number of complete occlusions was significantly reduced by intravenous administration of OM4 Fab (20 mg/kg) before or after mechanical injury to the vessel, without prolongation of bleeding time. Conclusion—Fab fragment of the monoclonal antibody OM4 effectively inhibits collagen induced platelet aggregation in vitro and ex vivo, and in vivo thrombosis in rats without prolonging bleeding time. Antibodies against GPVI may have therapeutic potential, inhibiting thrombosis without prolonging bleeding time.

MHC-dependent shaping of the inhibitory Ly49 receptor repertoire on NK cells: evidence for a regulated sequential model.

Engagement of MHC class I‐specific inhibitory receptors regulates natural killer (NK) cell development and function. Using both new and previously characterized anti‐Ly49 monoclonal antibodies, we comprehensively determined expression and co‐expression frequencies of four Ly49 receptors by NK cells from MHC‐congenic, MHC class I‐deficient, and Ly49A‐transgenic mice to study mechanisms that shape the inhibitory Ly49 repertoire. All Ly49 receptors were expressed on partially overlapping subsets. Significantly, in the absence of class I MHC, several receptor pairs were co‐expressed more frequently than predicted from a purely random expression model, indicating that biases independent of MHC class I underlie receptor co‐expression in some cases. MHC interactions were found to inhibit Ly49 co‐expression variably depending on the MHC allele and the receptor pair examined. These data extend previous evidence that interactions with MHC shape the repertoire. It was previously proposed that developing NK cells express Ly49 receptors sequentially and cumulatively, until self‐MHC specific receptors are expressed and inhibit new receptor expression. Fulfilling a major prediction of this model, we found that class I recognition by a Ly49A transgene expressed by all developing NK cells equivalently inhibited expression of endogenous self‐specific and nonself‐specific Ly49 receptors.

Ex vivo evaluation of anti-GPVI antibody in Cynomolgus monkeys: Dissociation between anti-platelet aggregatory effect and bleeding time

Recent progress in the understanding of thrombus formation has suggested an important role of glycoprotein (GP)VI. In contrast to its pivotal role in collagen-induced platelet activation, it has been suggested that its blockade does not induce massive bleeding tendency. To demonstrate the dissociation between inhibitory effect on platelet aggregation and bleeding by GPVI blockade, we examined the effects of Fab fragment of OM2, an anti-human GPVI monoclonal antibody on ex vivo collagen-induced platelet aggregation and skin bleeding time after intravenous injection in cynomolgus monkeys. In a dose-escalation study, OM2 potently (> 80%) inhibited collagen-induced platelet aggregation at the cumulative dose of 0.2 mg/kg with a slight prolongation of bleeding time (1.3 times baseline value). Furthermore, at 18.8 mg/kg, the highest dose tested, prolongation of bleeding time was still mild (1.9 times). In contrast, abciximab, Fab fragment of anti-GPIIb/IIIa antibody prolonged bleeding time by 5.0 times at 0.35 mg/kg, the lowest effective dose on platelet aggregation. In a pharmacodynamic study, a bolus injection of OM2 at 0.4 mg/kg produced potent inhibition of collagen-induced aggregation up to six hours after injection, showing longer half-life than that of abciximab. The injection of OM2 Fab did not induce thrombocytopenia and GPVI depletion in monkeys. These results suggest that blockade of GPVI by antibody can exert a potent inhibitory effect on collagen-induced platelet aggregation with a milder prolongation of bleeding time than blockade of GPIIb/IIIa. This study indicates that OM2 has the potential to be developed as a new class of therapeutic tool.